Methods of identifying, isolating and using odorant and aroma receptors

ABSTRACT

Provided here are new methods to identify specific families of mammalian odorant receptors for odorants or aroma, particularly indole and skatole malodors and their use in assays that may be used to discover compounds that modulate (blocking, enhancing, masking or mimicking compounds) their activity. Orphan mouse odorant receptors are identified from olfactory sensory neurons that respond to target compounds. The resulting receptors as well as their human counterparts can be screened in assays against test compounds to confirm their identity as odorant or aroma receptors, particularly malodor receptors and subsequently discover for example modulators that inhibit the perception of the malodor in humans.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.14/318,641 filed Jun. 28, 2014, which claims the benefit of U.S.provisional application 61/841,282, filed on Jun. 29, 2013 and of U.S.provisional application 61/921,664, filed on Dec. 30, 2013. The entirecontents of each of these applications are hereby incorporated byreference herein in their entirety.

SUBMISSION OF SEQUENCE LISTING

The Sequence Listing associated with this application is filed inelectronic format via EFS-Web and hereby incorporated by reference intothe specification in its entirety. The name of the text file containingthe Sequence Listing is 8950US_DIV_SequenceListing. The size of the textfile is 175 KB, and the text file was created on Jan. 19, 2018.

FIELD

The technical field is directed to odorant and aroma receptors andassays that can be used to identify odorant and/or aroma compounds andmore specifically inhibitors or counteractants of malodor compounds suchas indole, or skatole.

BACKGROUND

Olfaction is one of the most complex and poorly understood of humansensory systems. From olfactory receptor (OR) activation to perception,there are many steps that still require further investigation. If we canunderstand how the OR code for individual odorants and mixturestranslates into perception then we can exploit this knowledge to bringsignificant benefit in several areas. These areas include odormodulators like malodor counteractants that block the perception ofunpleasant odors, new flavor and fragrance ingredients that replacenon-biodegradable or toxic compounds, and odor enhancers that wouldlimit our reliance on difficult to source compounds from naturalsources. The ‘olfactory code’ combinatorial paradigm is centered on theobservation that any single OR may be activated by multiple odorants,and conversely most odorants are capable of activating several ORs. Inthe mouse genome there are 1,200 distinct ORs. Humans, by contrast, have˜400. In both cases, the repertoire of ORs is activated by manythousands of odorants in the world, and it is this combinatorialcomplexity that allows for the breadth of olfactory sensations we canperceive. However, odorants or ligands for only 95 mouse (˜8%) and 41human ORs (˜10%) have been identified as of 2014 using traditionaldeorphanization methods. In addition, the physiological relevance ofmost ligands for the human ORs, essentially identified in vitro, has notbeen tested.

Different OR de-orphanization methods have been described in theliterature [e.g. Touhara (2007) Deorphanizing vertebrate olfactoryreceptors: Recent advances in odorant-response assays Neurochem Int 51,132-139, Saito et al (2009) Odor coding by a Mammalian receptorrepertoire. Sci Signal 2, ra9, and Peterlin et al. (2014), The State ofthe Art of Odorant Receptor Deorphanization: a Report from theOrphanage, J Gen Physiol; 143(5): 527-42]. Many of these methods relyexclusively on cell-based assays where the OR is expressed innon-olfactory cells that are suitable for high-throughput screening.However, ORs are often retained in the endoplasmic reticulum of suchheterologous cells. Failing to traffic to the cell surface, the ORs arethus unable to interact with the odorant [Min et al. (2004) Endoplasmicreticulum degradation impedes olfactory G-protein coupled receptorfunctional expression. BMC Cell Biol 5, 34]. Thus, a systematic approachwhere hundreds to thousands of different cell lines, where each cellline possess a unique OR protein that can be assessed for odorantactivity, is not a suitable approach for comprehensive decoding of thecombinatoral interactions between odorants and ORs since many or most ofthe receptors do not function properly in such cell lines. There istherefore a need for new methods that can rapidly and reliably identifythe relatively small subset of ORs, within the entire repertoire of ORsthat exist in an organism, that are specifically activated or inhibitedby one or more odorants. There is a further need for a method to involvethe identification of ORs from the olfactory neurons themselves, wherethe ORs are presumed to be fully functional, thus circumventing thewell-known challenges of OR assays in non-olfactory cells.

Malodor compounds such as indole, skatole (3-methyl indole), andp-cresol generate unpleasant odors that arise for example from latrinesand other “bathroom” sources that contain fecal matter. Hence, malodorcounteractants that mask or reduce the perceived intensity or modify theperceived quality for example of human smell of the compounds aredesirable. Odorant receptors and more particularly malodor receptorshave a need to be identified. Receptors that bind to indole and skatolehave also been identified and compounds that bind to those receptorshave been discovered and reported as potential modulators of malodor.However, the rapid identification of the complete repertoire ofreceptors that bind to malodors, particularly indole or skatolereceptors continues to be desirable due to the numerous ORs that existin mammals. Assays that rely on new malodor receptors to identify newpotent compounds that bind to these receptors are further desired.

SUMMARY

Provided herein is a method of identifying olfactory receptors that areactivated by an odorant or aroma compound comprising:

-   -   a) dissociating an isolated olfactory epithelium containing        native olfactory neurons into single cells from a non-human        mammal species wherein each neuron expresses an olfactory        receptor;    -   b) loading the olfactory cells with an indicator dye that allows        for the measurement of odorant or aroma receptor binding        activity of the olfactory receptors;    -   c) contacting the olfactory receptors with odorant or aroma        compounds sequentially;    -   d) measuring changes of odorant or aroma-induced neuronal        activity;    -   e) isolating one or more olfactory neurons that were activated        by a odorant or aroma compound;    -   f) isolating or isolating and amplifying the mRNA of the        isolated olfactory receptors;    -   g) sequencing at least a portion of the transcriptome of the        mRNA by Next-Generation Sequencing; and    -   h) determining the identity of a group of olfactory receptors        selected from the group consisting of odorant and aroma        receptors by comparing the sequence of the transcriptome to a        reference genome sequence of the same species and other        vertebrate species.        Further provided herein is a method of identifying olfactory        receptors that are activated by a malodor compound comprising:    -   a) dissociating an isolated olfactory epithelium containing        native olfactory neurons into single cells from a non-human        mammal species wherein each neuron expresses an olfactory        receptor;    -   b) loading the olfactory cells with an indicator dye that allows        for the measurement of malodor receptor binding activity of the        olfactory receptors;    -   c) contacting the olfactory receptors with malodor compounds        sequentially;    -   d) measuring changes of odorant-induced neuronal activity;    -   e) isolating one or more olfactory neurons that were activated        by a malodor compound;    -   f) isolating or isolating and amplifying the mRNA of the        isolated olfactory receptors;    -   g) sequencing at least a portion of the transcriptome of the        mRNA by Next-Generation Sequencing; and    -   h) determining the identity of a group of malodor olfactory        receptors by comparing the sequence of the transcriptome to a        reference genome sequence of the same species and other        vertebrate species.

Further provided herein is an isolated nucleic acid sequence having atleast 60% sequence identity with a nucleic acid sequence selected fromthe group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ IDNO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ IDNO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45,SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO:55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ IDNO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83and SEQ ID NO: 85.

Also provided herein is an isolated nucleic acid sequence as describeabove which encodes a polypeptide having at least 60% sequence identifywith a polypeptide having an amino acid sequence selected from the groupconsisting of: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8,SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO:28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ IDNO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56,SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO:66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ IDNO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84 andSEQ ID NO: 86.

Further provided herein is an isolated polypeptide comprising an aminoacid sequence having at least 60% sequence identify with an amino acidsequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO:4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO:14, SEQ ID NO: 16, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ IDNO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52,SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO:62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ IDNO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQID NO: 82, SEQ ID NO: 84 and SEQ ID NO: 86.

Still yet further provided is a cell that is recombinantly modified toexpress a polypeptide, described above.

Further provided are assays for identifying compounds that bind toindole and/or skatole odorant receptors. In particular, provided hereinis a method for identifying a compound that blocks, inhibits, modulates,and/or enhances the activity of an olfactory receptor that is activatedby a compound selected from the group consisting of indole and skatolecomprising

-   -   a) contacting the receptor, or a chimera or fragment with a        compound;    -   b) assaying whether the compound has an effect on the activity        of the receptor;        -   wherein the receptor is a polypeptide described above.

In one embodiment the activity of the compound is determined bycomparing its binding to that of indole and/or skatole. In anotherembodiment, the receptor is contacted with a compound in the presence ofskatole and/or indole under conditions that allow for the binding of thecompound along with skatole and/or indole to the receptor.

In a further embodiment, provided herein, when a functional assay isused to measure binding activity, the step of measuring a signalingactivity of receptors provided herein may comprise detecting a change inthe level of a second messenger. In another embodiment, the measurementof a signalling activity is provided wherein the step of measuring asignaling activity comprises the measurement of guanine nucleotidebinding/coupling or exchange, adenylate cyclase activity, cAMP, ProteinKinase C activity, Protein Kinase A activity phosphatidylinosotolbreakdown, diacylglycerol, inositol triphosphate, intracellular calcium,calcium flux, arachidonic acid, MAP kinase activity, tyrosine kinaseactivity, melanophore assay, receptor initialization assay, FRET, BRET,or reporter gene expression. In a particular embodiment provided herein,the measuring of signaling activity comprises using a fluorescence orluminescence assay. Fluorescence and luminescence assays may comprisethe use of Ca²⁺ sensitive fluorophores including Fluo-3, Fluo-4 or Furadyes (Molecular Probes); Calcium 3 assay kit family (Molecular Devices)and aequorin.

DETAILED DESCRIPTION OF THE DRAWINGS

FIG. 1 displays a schematic overview of the malodor receptorde-orphanization process

FIG. 2 displays Ca²⁺ imaging traces that are shown for 6 independentolfactory sensory neurons specifically activated by both indole andskatole (50 μM each).

FIG. 3 shows malodor receptor genes identified using the proceduresoutlined herein.

FIGS. 4A and 4B display human and additional mouse malodor receptorswithin the Olfr740 (4A) and Olfr665 (4B) families identified using theprocedures outlined herein

FIGS. 5A to 5E show the indole and skatole activity of mouse ORsOlfr743, Olfr746, and Olfr740 in HEK293T cells.

FIGS. 6A to 6J show the indole and skatole activity of human OR52N2,OR11G2, OR5AC2, OR4C15, OR8S1, OR11H6 and OR11H4 and mouse Olfr665 andOlfr740 in HEK293T cells.

FIG. 7 shows the inhibition of indole receptor Olfr743 activity inHEK293T cells by a test compound.

FIG. 8 shows the pairwise amino acid identities for the mouse Olfr740family and their predicted human orthologs.

FIG. 9 shows the pairwise amino acid identities for the mouse Olfr665family and their predicted human orthologs.”

DETAILED DESCRIPTION

For the descriptions herein and the appended claims, the use of “or”means “and/or” unless stated otherwise. Similarly, “comprise,”“comprises,” “comprising” “include,” “includes,” and “including” areinterchangeable and not intended to be limiting.

It is to be further understood that where descriptions of variousembodiments use the term “comprising,” those skilled in the art wouldunderstand that in some specific instances, an embodiment can bealternatively described using language “consisting essentially of” or“consisting of”

Definitions

The following terms have the meanings ascribed to them unless specifiedotherwise.

“OR” refers to one or more members of a family of G protein-coupledreceptors that are expressed in olfactory cells. Olfactory receptorcells can also be identified on the basis of morphology or by theexpression of proteins specifically expressed in olfactory cells. ORfamily members may have the ability to act as receptors for olfactorytransduction.

“Indole” and/or “skatole OR” refers to a member of the family of Gprotein-coupled receptors that is expressed in an olfactory cell, whichreceptors bind and/or are activated by indole and/or skatole in abinding or activity assay for identifying ligands that bind and modulateGPCRs by activating, inhibiting or enhancing their activity. Such assaysare described below. Indole and/or skatole receptors herein will includefragments, variants, including synthetic and naturally occurring, andchimeras that respond to or bind indole and/or skatole.

“OR” nucleic acids encode a family of GPCRs with seven transmembraneregions that have “G protein-coupled receptor activity,” e.g., they maybind to G proteins in response to extracellular stimuli and promoteproduction of second messengers such as IP3, cAMP, cGMP, and Ca²⁺ viastimulation of enzymes such as phospholipase C and adenylate cyclase.

“OR” polypeptides are considered as such if they pertain to the7-transmembrane-domain G protein-coupled receptor superfamily encoded bya single ˜1 kb long exon and exhibit characteristic olfactoryreceptor-specific amino acid motifs. The predicted seven domains arecalled “transmembrane” or “TM” domains TM I to TM VII connected by threepredicted “internal cellular loop” or “IC” domains IC I to IC III, andthree predicted “external cellular loop” or “EC” domains EC I to EC III.The motifs are defined as, but not restricted to, the MAYDRYVAIC motifoverlapping TM III and IC II, the FSTCSSH motif overlapping IC III andTM VI, the PMLNPFIY motif in TM VII as well as three conserved Cresidues in EC II, and the presence of highly conserved GN residues inTM I [Zhang and Firestein (2002), The Olfactory Receptor GeneSuperfamily of the Mouse. Nature Neuroscience: 5(2):124-33; Malnic etal., The Human Olfactory Receptor Gene Family: PNAS: 101(8):2584-9].

The “N terminal domain” region starts at the N-terminus and extends to aregion close to the start of the first predicted transmembrane region.“Transmembrane domain,” which comprises the seven predicted“transmembrane regions,” refers to the domain of OR polypeptides thatlies within the plasma membrane, and may also include the correspondingcytoplasmic (intracellular) and extracellular loops. The seventransmembrane regions and extracellular and cytoplasmic loops can beidentified using standard methods, as described in Kyte & Doolittle, J.Mol. Biol., 157:105-32 (1982), or in Stryer. The general secondary andtertiary structure of transmembrane domains, in particular the seventransmembrane domains of G protein-coupled receptors such as olfactoryreceptors, are known in the art. Thus, primary structure sequence can bedesigned or predicted based on known transmembrane domain sequences, asdescribed in detail below. These transmembrane domains are useful for invitro ligand-binding assays, both soluble and solid phase.

The phrase “functional assay” in the context of assays for testingcompounds that modulate OR family member mediated olfactory transductionincludes the determination of any parameter that is indirectly ordirectly under the influence of the receptor, e.g., functional, physicaland chemical effects. It includes ligand binding, changes in ion flux,membrane potential, current flow, transcription, G protein binding, GPCRphosphorylation or dephosphorylation, signal transductionreceptor-ligand interactions, second messenger concentrations (e.g.,cAMP, cGMP IP3, or intracellular Ca²⁺), assays performed in vitro, invivo, and ex vivo. It also includes other physiological effects such asincreases or decreases of neurotransmitter or hormone release. Includedherein are functional assays for a compound that increases or decreasesa parameter that is indirectly or directly under the influence of an ORfamily member, e.g., functional, physical and chemical effects. Suchfunctional assays or effects can be measured by any means known to thoseskilled in the art, e.g., changes in spectroscopic characteristics(e.g., fluorescence, luminescence, absorbance, refractive index),hydrodynamic (e.g., shape), chromatographic, solubility properties,patch clamping, voltage-sensitive dyes, whole cell currents,radioisotope efflux, inducible markers, oocyte OR gene expression;tissue culture cell OR expression; transcriptional activation of ORgenes; ligand-binding assays; voltage, membrane potential andconductance changes; ion flux assays; changes in intracellular secondmessengers such as cAMP, cGMP, and inositol triphosphate (IP3); changesin intracellular calcium levels; neurotransmitter release, and the like.

“Inhibitors,” “blockers,” “activators,” “counteractants” and“modulators” of OR genes or proteins are used interchangeably to referto inhibitory, activating, or modulating molecules identified using exvivo, in vitro and in vivo assays for olfactory transduction, e.g.,ligands, agonists, antagonists, inverse agonists and their homologs andmimics. Inhibitors and blockers are compounds that, e.g., bind to,partially or totally block stimulation, decrease, prevent, delayactivation, inactivate, desensitize, or down regulate olfactorytransduction, e.g., antagonists. Activators are compounds that, e.g.,bind to, stimulate, increase, open, activate, facilitate, enhanceactivation, sensitize, or up regulate olfactory transduction, e.g.,agonists. Modulators include compounds that, e.g., alter the interactionof a receptor with: extracellular proteins that bind activators orinhibitors (e.g., odorant-binding proteins, lipocalin and other membersof the hydrophobic carrier family); G proteins; kinases (e.g., homologsof rhodopsin kinase and beta adrenergic receptor kinases that areinvolved in deactivation and desensitization of a receptor); andarrestins, which also deactivate and desensitize receptors. Modulatorscan include genetically modified versions of OR family members, e.g.,with altered activity, as well as naturally occurring and syntheticligands, antagonists, agonists, small chemical molecules and the like.Such assays for inhibitors and activators include, e.g., expressing ORfamily members in cells or cell membranes, applying putative modulatorcompounds, in the presence or absence of flavor or fragrance molecules,e.g., perfumery raw materials, perfume formulations, or malodors, andthen determining the functional effects on olfactory transduction, asdescribed above. Samples or assays comprising OR family members that aretreated with a potential activator, inhibitor, or modulator are comparedto control samples without the inhibitor, activator, or modulator toexamine the extent of modulation. Control samples (untreated withmodulators) are assigned a relative OR activity value of 100%.Inhibition of an OR is achieved when the OR activity value relative tothe control is about 80%, optionally 50% or 25-0%. Activation of an ORis achieved when the OR activity value relative to the control is 110%,optionally 150%, optionally 200-500%, or 1000-3000% higher.

The terms “purified,” “substantially purified,” and “isolated” as usedherein refer to the state of being free of other, dissimilar compoundswith which the compound of the invention is normally associated in itsnatural state, so that the “purified,” “substantially purified,” and“isolated” subject comprises at least 0.5%, 1%, 5%, 10%, or 20%, andmost preferably at least 50% or 75% of the mass, by weight, of a givensample. In one preferred embodiment, these terms refer to the compoundof the invention comprising at least 95% of the mass, by weight, of agiven sample.

As used herein, the term “isolated,” when referring to a nucleic acid orpolypeptide refers to a state of purification or concentration differentthan that which occurs naturally in the mammalian, especially human,body. Any degree of purification or concentration greater than thatwhich occurs naturally in the body, including (1) the purification fromother naturally-occurring associated structures or compounds, or (2) theassociation with structures or compounds to which it is not normallyassociated in the body are within the meaning of “isolated” as usedherein The nucleic acids or polypeptides described herein may beisolated or otherwise associated with structures or compounds to whichthey are not normally associated in nature, according to a variety ofmethods and processed known to those of skill in the art.

As used herein, the terms “amplifying” and “amplification” refer to theuse of any suitable amplification methodology for generating ordetecting recombinant of naturally expressed nucleic acid, as describedin detail, below. For example, the invention provides methods andreagents (e.g., specific degenerate oligonucleotide primer pairs) foramplifying (e.g., by polymerase chain reaction, PCR) naturally expressed(e.g., genomic or mRNA) or recombinant (e.g., cDNA) nucleic acids of theinvention in vivo, ex vivo or in vitro.

The term “7-transmembrane receptor” means a polypeptide belonging to asuperfamily of transmembrane proteins that have seven domains that spanthe plasma membrane seven times (thus, the seven domains are called“transmembrane” or “TM” domains TM I to TM VII). The families ofolfactory and certain taste receptors each belong to this super-family.7-transmembrane receptor polypeptides have similar and characteristicprimary, secondary and tertiary structures, as discussed in furtherdetail below.

The term “nucleic acid” or “nucleic acid sequence” refers to adeoxy-ribonucleotide or ribonucleotide oligonucleotide in either single-or double-stranded form. The term encompasses nucleic acids, i.e.,oligonucleotides, containing known analogs of natural nucleotides. Theterm also encompasses nucleic-acid-like structures with syntheticbackbones. Unless otherwise indicated, a particular nucleic acidsequence also implicitly encompasses conservatively modified variantsthereof (e.g., degenerate codon substitutions or single nucleotidepolymorphisms) and complementary sequences, as well as the sequenceexplicitly indicated. Specifically, degenerate codon substitutions maybe achieved by generating, e.g., sequences in which the third positionof one or more selected codons is substituted with mixed-base and/ordeoxyinosine residues.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers and non-naturally occurring amino acid polymer. The term“heterologous” when used with reference to portions of a nucleic acidindicates that the nucleic acid comprises two or more subsequences thatare not found in the same relationship to each other in nature. Forinstance, the nucleic acid is typically recombinantly produced, havingtwo or more sequences from unrelated genes arranged to make a newfunctional nucleic acid, e.g., a promoter from one source and a codingregion from another source. Similarly, a heterologous protein indicatesthat the protein comprises two or more subsequences that are not foundin the same relationship to each other in nature (e.g., a fusionprotein).

As used herein, “recombinant” refers to a polynucleotide synthesized orotherwise manipulated in vitro (e.g., “recombinant polynucleotide”), tomethods of using recombinant polynucleotides to produce gene products incells or other biological systems, or to a polypeptide (“recombinantprotein”) encoded by a recombinant polynucleotide. “Recombinant” meansalso encompass the ligation of nucleic acids having various codingregions or domains or promoter sequences from different sources into anexpression cassette or vector for expression of, e.g., inducible orconstitutive expression of a fusion protein comprising a translocationdomain of the invention and a nucleic acid sequence amplified using aprimer of the invention.

The term “expression vector” refers to any recombinant expression systemfor the purpose of expressing a nucleic acid sequence of the inventionin vitro or in vivo, constitutively or inducibly, in any cell, includingprokaryotic, yeast, fungal, plant, insect or mammalian cell. The termincludes linear or circular expression systems. The term includesexpression systems that remain episomal or integrate into the host cellgenome (e.g. stable expression). The expression systems can have theability to self-replicate or not, i.e., drive only transient expressionin a cell. The term includes recombinant expression “cassettes” whichcontain only the minimum elements needed for transcription of therecombinant nucleic acid.

By “host cell” is meant a cell that contains an expression vector andsupports the replication or expression of the expression vector. Hostcells may be prokaryotic cells such as E. coli, or eukaryotic cells suchas yeast, insect, amphibian, or mammalian cells such as CHO, HeLa,HEK-293, and the like, e.g., cultured cells, explants, and cells invivo.

In one embodiment provided herein is an isolated nucleic acid sequencehaving at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% sequenceidentity with a nucleic acid sequence selected from the group consistingof SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9,SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 27, SEQ ID NO:29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ IDNO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57,SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO:67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ IDNO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83 and SEQ ID NO: 85.

In one embodiment provided herein is an isolated nucleic acid sequenceas described above which encodes a polypeptide having at least 60%, 65%,70%, 75%, 80%, 85%, 90%, 95% or 98% sequence identity with a polypeptidehaving an amino acid sequence selected from the group consisting of: SEQID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 28, SEQ ID NO: 30,SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO:40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ IDNO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68,SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO:78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84 and SEQ ID NO: 86.

In a further embodiment provided herein is an isolated polypeptidecomprising an amino acid sequence having at least 60%, 65%, 70%, 75%,80%, 85%, 90%, 95% or 98% sequence identity with an amino acid sequenceselected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ IDNO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ IDNO: 16, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44,SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO:54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ IDNO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82,SEQ ID NO: 84 and SEQ ID NO: 86.

A method enables one skilled in the art to rapidly identify therelatively small subset of odorant receptors (ORs), within the totalpopulation of 1,200 ORs that exist in the rodent genome, whose humangenetic counterparts likely encode a specific odor characteristic inhumans. FIG. 1 is a schematic overview of the method. Intact mouseolfactory epithelium (OE) tissue is dissociated by mechanical andenzymatic treatment into single cells. The resulting individualolfactory sensory neurons (OSNs) are plated onto glass coverslips andloaded with a calcium indicator dye (e.g., Fura-2) to detect cellactivity. The coverslips are then placed in a perfusion chamber andbathed in a physiological salt solution. Malodorous test odorants arediluted in physiological saline and perfused over the cells. OSNactivity is then detected by monitoring the changes in intracellularcalcium flux via a change in Fura-2 fluorescence and a fluorescentmicroscope. Importantly, in some instances, only one or two of ˜1200possible OR genes are expressed in a given OSN. This means that theactivity of a given OSN is driven exclusively by a single type, or twotypes, of OR protein(s) expressed on the cell surface. A semi-automatedcalcium imaging system with a finely controlled odorant injection systemand a movable stage allows for monitoring of >1,500 OSNs in a singleexperiment. With ˜1,200 OR genes in mouse, this throughput ensures arepresentative sampling of the entire OR repertoire. One or more OSNsthat respond to one or more test odorant are collected using a glassmicropipette and displaced in a microtube for subsequent RNA extraction,amplification, and Next-Generation Sequencing (NGS) of OSNtranscriptomes. NGS-based transcriptome sequencing from very smallamounts of starting material (<100 cells) has been reported in theliterature. However, it has not yet been applied for the specificpurpose of G-Protein Coupled Receptor (GPCR) deorphanization to the bestof our knowledge; in particular olfactory receptors. Using the methodsdescribed herein, multiple OSNs can be pooled in the same microtube ifdesired. Raw sequence reads are then mapped to a reference genome,visually inspected for proper alignment and annotation, and analyzed forfalse-positive mapping due to, for example, genome repeat sequences. Theresult is a list of candidate mouse ORs that are likely activated by thesame malodorous test odorant(s) used to screen mouse OSNs. The candidatemouse OR protein sequences are then used to query (e.g., BLASTP) publicmouse genome databases to obtain a full list of candidate mouse ORsequences. These candidates are related by sequence homology (i.e.paralogous) to the NGS-derived OR sequences originally identified fromthe neurons that were activated by indole and/or skatole. The full listof mouse paralogs (NGS and BLASTP-derived) are then used to query publichuman genome databases to obtain a list candidate human OR sequencesthat are related (i.e. orthologous) to the mouse OR sequences. Theresult is a complete list of candidate mouse and human ORs that arelikely activated by the test odorants. To confirm the activity of theresulting candidate ORs genes, cDNAs of said genes can be cloned intoavailable expression vectors and transfected into cultured cells thatare amenable to high-throughput screening (e.g. HEK293T). The resultingcell lines, each containing a recombinantly expressed candidate OR cDNA,can then be screened in a cell-based activity assay with the testodorants used in calcium imaging and other structurally- ororganoleptically-related odorants.

In a particular embodiment olfactory epithelium containing nativeolfactory neurons from a non-human mammal (e.g., mouse) are isolated anddissociated into single cells according to for example Araneda et al.(2004), A Pharmacological Profile of the Aldehyde Receptor Repertoire inRat Olfactory Epithelium. Journal of Physiology: 555:743-756. In aparticular embodiment, each neuron expresses only one or two receptorsat a time. The olfactory tissue used to generate olfactory neurons is,in one embodiment, from a non-human mammal that has publically availabledata concerning the receptors and associated sequences. This includesmouse, rat and hamster tissue. The dissociation protocol should beoptimized in order to ensure high rate of surviving recordable neurons.In a particular embodiment a minimum of approximately 1,500 distinctsensory neurons are recorded.

In a further embodiment, the indicator is selected from a fluorescentcalcium indicator dye, a calcium indicator protein, a fluorescent cAMPindicator, a cAMP response element (CRE) mediated reporter protein, abiochemical cAMP HTRF assay, a beta-arrestin assay, or anelectrophysiological recording. Particularly, a calcium indicator dye isselected that can be used to monitor the activity of olfactory receptorsexpressed on the membrane of the olfactory neurons (e.g., Fura-2 AM).

In a particular embodiment, compounds are screened sequentially and theodorant-dependant changes in calcium dye fluorescence are measured usinga fluorescent microscope or fluorescent-activated cell sorter (FACS).

As an example, olfactory neurons are isolated after screening with oneor more malodor compounds using either a glass microelectrode attachedto a micromanipulator or a FACS machine. More particularly at least 1neuron is isolated. Mouse olfactory sensory neurons are screened by Ca²⁺imaging similar to procedures previously described (Malnic et al., 1999;Areneda et al., 2004). Particularly, a motorized movable microscopestage is used to increase the number of cells that can be screened to atleast 1,500 per experiment. Since there are approximately 1,200different olfactory receptors in the mouse and each olfactory sensoryneuron expresses only 1 or 2 of 1,200 olfactory receptor genes, thisscreening capacity will cover virtually the entire mouse odorantreceptor repertoire. In other words, the combination of calcium imagingfor high-throughput olfactory sensory neuron screening leads to theidentification of nearly all of the odorant receptors that respond to aparticular profile of odorants. In a particular aspect, odorants thatrespond to both indole and skatole, two common latrine malodor compoundscan be isolated.

For calcium imaging of olfactory neurons, the main olfactory epitheliummay be dissected from a mouse before neuronal dissociation. Dissectedolfactory epithelium may then be transferred to a dissociation bufferfor mechanical and enzymatic dissociation. Dissociated neurons may thenbe seeded onto a coverslip allowing the screening of thousands of cellsby fluorescence microscopy and the cells may be loaded with a calciumsensitive dye (Fura-2 AM) for example for about 30 minutes at 31° C. andtransferred onto the microscope ready for screening. Cells arestimulated by perfusing diluted solutions of odorants (in physiologicalsaline) over the dissociated olfactory neurons. The rare cells thatrespond to the malodor compound are identified by for examplestimulating the receptors with 50 μm of the malodor compounds and thenby monitoring the intracellular Ca²⁺ flux indicated by changes in Fura-2fluorescence. After analysis, responding cells may be retrieved from aglass coverslip with a suction micropipette. Isolated cells are thenpooled into one sample for subsequent identification of the odorantreceptor genes expressed as mRNA in the responding cells.

In a particular embodiment, the mRNA of olfactory neurons are purifiedand amplified according to the method generally described in Marko, N.F., et al., (2005) A robust method for the amplification of RNA in thesense orientation. BMC genomics, 6, 27; doi:10.1186/1471-2164-6-27(Eberwine method). At least a portion of the transcriptome (up to andincluding the entire transcriptome) is sequenced using Next-GenerationSequencing (NGS) or hybridized to known genes using Microarraytechnologies. NGS is generally discussed and described in Metzker, M. L.(2010). Sequencing technologies—the next generation. Nature reviews.Genetics, 11(1), 31-46; doi:10.1038/nrg262. In a particular embodiment,a minimum of 5 neurons presenting the same response profile are pooledand the mRNA are amplified by two consecutive rounds of in vitrotranscription (IVT). The mRNA is released by cell lysis immediatelyafter picking; no DNAse and no purification steps are carried out. Theamplification may be done according to MesageAmpll aRNA kit (Ambion,AMA1751) with the following parameters: two rounds of consecutive 14hour long IVT.

In a further embodiment, the identity of a group or gene family ofmalodor olfactory receptors is determined (e.g., up to as many as thenumber of neurons picked) by comparing the results of the NGS to areference genome sequence of the same species. Particularly, theputative malodor receptors will be the most highly abundant mRNA in theolfactory neuron-derived NGS sample or present in more than oneindependent biological replicate. Because of the combinatorial nature ofthe olfactory code (one compound activates many ORs and one OR can beactivated by many compounds), pooling several neurons activated by givencompounds allows the retrieval of virtually all of the receptorsresponsible for the perception of these molecules in a single NGSexperiment. Pooling functionally similar neurons thus greatly improvesthe deorphanization throughput and speed.

Standard bioinformatics tools are then used to identify the most closelyrelated human odorant receptor(s) to other putative mammalian(non-human) malodor receptor(s) under the assumption that homologousreceptor sequences retain similar function. One study demonstrated that˜82% of orthologous OR gene pairs responded to a common ligand,suggesting that as many as ˜18% of orthologous gene pairs may respond toa different ligand; further, 33% of paralogous OR gene pairs respondedto a common ligand, suggesting that at least ˜67% of paralogous pairsmay respond to a different ligand [Adipietro et al. (2012) FunctionalEvolution of Mammalian Odorant Receptors. PLoS Genet 8(7): e1002821.doi:10.1371/journal.pgen. 1002821]. Default parameters of BLASTP and/orBLASTN algorithm may be used.

The human or non-human mammalian malodor receptor may be adapted to afunctional assay that can be used to identify compounds that mimic,block, modulate, and/or enhance the activity of a malodor compound. Inparticular the assay may be a cell assay and the method for identifyingcompounds may be a high-throughput screening assay. More particularly,provided herein are receptor-based assays adaptable for high-throughputscreening of receptors with compound libraries for the discovery ofmodulating compounds (e.g., blocking, enhancing, and masking).

In a particular embodiment, malodor receptor gene sequences areidentified from indole and skatole-sensitive cells as follows: Pooledneurons are heated to 75° C. for 10 minutes to break the cell membraneand render their mRNA available for amplification. This amplificationstep is important when applying NGS technologies with limited amount ofstarting material, typically between 1 to 15 cells. A linearamplification according to the Eberwine method (IVT) ensures themaintenance of the relative transcription levels of expressed genes. Twoconsecutive overnight (14 h) rounds of in vitro transcription are usedto yield sufficient amounts of cRNA; Amplified cRNA is then used togenerate an Illumina HiSeq cDNA library. The resulting short sequencesof typically 75-150 base pairs (commonly referred to as “reads”) arealigned against the reference genome of the mouse (such as UCSC versionmm9 or mm10) in order to build the full transcriptome of these cells.Quantitative analysis of the transcriptome data yields a list oftranscribed odorant receptor genes and their respective expressionlevels. Odorant receptor genes that show the most abundant levels ofmRNA (most abundant “reads”) or are present in more than one replicateexperiment are considered putative indole and skatole receptors.

The predicted mouse OR genes are then used to mine the latest versionsof both the mouse and human genome databases in order to identify themost closely related receptors (i.e., highest sequence similarity) inmouse (paralogous genes) and in human (orthologous genes). This processmay be performed using the BLAST search algorithm (publically availableat the NCBI website), a sequence similarity search tool, where everyputative gene sequence previously obtained from the initialtranscriptome analysis is used as a query sequence. The newly identifiedgenes identified from this data mining process are also considered aspotential malodor receptors under the assumption that paralogous andorthologous genes are highly likely to possess similar activities.

In a particular embodiment, pairwise comparison of sequence homology iscarried out to identify closely related receptors in mouse and humansusing the following iterative scheme:

Step Query sequence BLASTN/BLASTP Result 1. Mouse candidate 1 → Mouseparalog 1 and human ortholog 1 2. Mouse paralog 1 → Human ortholog 2 3.Human ortholog 1 → Human paralog 1 4. Human ortholog 2 → Human paralog 2Paralog = homolog in same species Ortholog = homolog in other species

Paralogous genes are then aligned using a multiple alignment tool inorder to generate a phylogenetic tree. Functional in vitro data can beinterpreted in the light of such a phylogenetic relationship betweenclosely related but distinct receptors. This step is essential in theidentification of complete OR gene families that respond, to varyingdegrees, to the test compounds, for example indole and skatole.

To complete the deorphanization process, the candidate OR genes arefurther expressed in vitro for confirmation of activity against thecompounds used to isolate the olfactory sensory neurons and otherstructurally-related compounds of interest. In one embodiment, tocomplete the deorphanization process, the candidate OR genes are furtherexpressed in vitro for confirmation of activity against the compoundsinitially used to isolate said olfactory sensory neurons. The samecandidate OR genes expressed in vitro are further screened with otherstructurally-related compounds of interest to identify, e.g.,activators, inhibitors, or modulators of the receptor.

Using the process and method described herein the following receptorshave been identified directly by sequencing (NGS) the transcriptome ofolfactory neurons responding to indole and skatole malodors (mouse ORs).Human indole and skatole receptors with the highest degree of amino acididentity to the mouse ORs were subsequently identified by searchingavailable genome databases (Table 1). Mouse receptor Olfr740 and Olfr665were initially identified through the NGS method described herein afterisolating or “picking” responding cells after exposure to both indoleand skatole. Corresponding human homologous receptors, OR11G2 and OR52N2respectively, were further identified by amino acid sequence similaritycomparisons. As demonstrated in examples 4 and 5, the candidate mousereceptors Olfr740 and Olfr665 and their human counterparts OR11G2 andOR52N2 were activated in vitro by indole and by skatole and are thusdescribed as indole and/or skatole receptors. Similarly, the candidatemouse receptors Olfr 746, Olfr745, Olfr207, Olfr1211 and Olfr257 led toidentification of the human receptors OR11H4, OR11H6, OR5AC2, OR4C15 andOR8S1, respectively, following the same steps. These human OR genes alsoresponded to both indole and skatole in vitro. Taken together, seven outof seven tested putative human receptors identified by the methoddescribed herein were confirmed as indole and skatole receptors furthersupporting the method for efficient human indole and skatole receptoridentification.

This approach to odorant receptor deorphanization has several majoradvantages over previously established single cell RT-PCR methods.First, by pooling multiple neurons sharing similar binding properties toodors and/or aromas, a unique mRNA sequencing experiment (NGS)identifies virtually all the receptors that are activated by the targetmalodor compounds. Therefore the throughput is higher than what waspreviously achieved. Second, because multiple cells are pooled into onesample, multiple ORs for a particular compound are revealed in a singleexperiment. In a particular embodiment, the selection of genes through acomprehensive comparison of replicate samples across experiments. Third,NGS does not require the use of PCR primers specific to an OR. NGS alsodoes not require the use of degenerate primers specific to ORs, whichare problematic and often lead to false positives due to non-linear ornon-specific PCR amplification. In particular, since OR coding sequenceslie within a single exon, sample contamination with genomic DNA caneasily lead to an aspecific amplification of OR gene sequences. Fourth,RT-PCR analysis is difficult to perform on pooled samples because of theinherent false positive rate. Single cell mRNA hybridization experimentshave been performed using of high-density DNA microarray chips. However,this approach is generally less sensitive than NGS and is furtherrestricted to known genes for which corresponding DNA probes need to besynthesized. Hence, the use of NGS is significantly advantageous torapidly identify OR and ultimately results in a more accurate selectionof candidate receptors compared to the standard (e.g., RT-PCR andmicroarray) approaches.

In a further embodiment, mouse receptors identified from isolatedolfactory neurons that respond to both indole and skatole are modifiedat their N-terminus with a short polypeptide sequence (e.g., bovinerhodopsin receptor—Rho, or Flag), transiently expressed in HEK 293Tcells, and stimulated separately with indole and skatole to confirmtheir identity as bona fide indole/skatole receptors. Co-expression ofthe human G alpha subunit Gα_(olf) (SEQ ID NO: 21) activates the Gstransduction pathway that leads to an internal cAMP increase uponbinding to the appropriate ligand. The results confirm the identity ofOlfr740 (SEQ ID NO: 1 and SEQ ID NO: 2), Olfr743 (SEQ ID NO: 5 and SEQID NO: 6), and Olfr746 (SEQ ID NO: 37 and SEQ ID NO: 38) asindole-skatole receptors.

In another aspect, human receptor OR52N2 (SEQ ID NO: 11 and SEQ ID NO:12) was identified because of its sequence similarity to mouse Olfr665(SEQ ID NO: 9 and SEQ ID NO: 10), an indole/skatole receptor isolatedfrom olfactory neurons responding to both indole and skatole (Example6). Human receptor OR11G2 (SEQ ID NO: 13 and SEQ ID NO: 14) wasidentified because of its sequence similarity to mouse Olfr740 (SEQ IDNO: 1 and SEQ ID NO: 2), an indole/skatole receptor isolated fromolfactory neurons responding to both indole and skatole (Example 6).Human receptor OR5AC2 (SEQ ID NO: 75 and SEQ ID NO: 76) was identifiedbecause of its sequence similarity to mouse Olfr207 (SEQ ID NO: 77 andSEQ ID NO: 78), an indole/skatole receptor isolated from olfactoryneurons responding to both indole and skatole (Example 6). Humanreceptor OR4C15 (SEQ ID NO: 79 and SEQ ID NO: 80) was identified becauseof its sequence similarity to mouse Olfr1211 (SEQ ID NO: 81 and SEQ IDNO: 82), an indole/skatole receptor isolated from olfactory neuronsresponding to both indole and skatole (Example 6). Human receptor OR8S1(SEQ ID NO: 83 and SEQ ID NO: 84) was identified because of its sequencesimilarity to mouse Olfr257 (SEQ ID NO: 85 and SEQ ID NO: 86), anindole/skatole receptor isolated from olfactory neurons responding toboth indole and skatole (Example 6). Mouse receptor Olfr665 (SEQ ID NO:9 and SEQ ID NO: 10) was identified directly from NGS data from isolatedolfactory neurons responding to both indole and skatole (Example 6).Mouse receptor Olfr740 (SEQ ID NO: 1 and SEQ ID NO: 2) was identifieddirectly from NGS data from isolated olfactory neurons responding toboth indole and skatole (Example 6). Mouse receptor Olfr736 (SEQ ID NO:27 and SEQ ID NO: 28) was identified because of its sequence similarityto mouse Olfr740 (SEQ ID NO: 1 and SEQ ID NO: 2), an indole/skatolereceptor isolated from olfactory neurons and failed to respond to indoleor skatole likely because of lack of cell surface expression. Mousereceptor Olfr747 (SEQ ID NO: 39 and SEQ ID NO: 40) and Olfr748 (SEQ IDNO: 41 and SEQ ID NO: 42) were also identified because of their sequencesimilarity to mouse Olfr740 (SEQ ID NO: 1 and SEQ ID NO: 2) andexceptionally failed to respond to indole or skatole despite proper cellsurface expression as evaluated by immunostaining assays. Thisunderlines the diversifying nature of the olfactory receptors wherehighly similar sequences do not always share the same response profile.

The receptors are modified with the Rho sequence and stably expressed inHEK 293T cells. Co-expression of the human G alpha subunit Gα₁₅activates the Gq transduction pathway that leads to an internal Ca²⁺increase upon binding to the appropriate ligand.

The above process and the results obtained so far serve to validate theprocess for rapid and reliable identification of mammalian odorantreceptors for malodor compounds.

Still yet further provided is a cell that is recombinantly modified toexpress a polypeptide described above.

Further provided are assays for identifying compounds that bind toindole and/or skatole odorant receptors. In particular, provided hereinis a method for identifying a compound that blocks, inhibits, modulates,and/or enhances the activity of an olfactory receptor that is activatedby a compound selected from the group consisting of indole and skatolecomprising

-   -   a) contacting the receptor, or a chimera or fragment thereof        with a compound;    -   b) assaying whether the compound has an effect on the activity        of the receptor;        -   wherein the receptor is a polypeptide described above.

In one embodiment the activity of the compound is determined bycomparing its binding to that of indole and/or skatole. In anotherembodiment, the receptor or a chimera or fragment thereof is contactedwith a compound in the presence of skatole and/or indole underconditions that allow for the binding of the compound along with skatoleand/or indole to the receptor.

In a further embodiment, a compound is contacted to a receptor, or achimera or fragment thereof that is activated by a compound selectedfrom the group consisting of indole and skatole wherein the receptor, ora chimera or fragment thereof is expressed in a cell that isrecombinantly modified to express the polypeptide.

The activity of the compound can be determined using in vivo, ex vivo,in vitro and synthetic screening systems, which typically allow forscreening of large libraries of compounds, containing as many as 10,000sof compounds or mixtures of compounds.

In another embodiment, the contacting is performed with liposomes orvirus-induced budding membranes containing the polypeptides describedherein.

In another embodiment, the methods for identifying compounds that bindto the receptors that bind to skatole and/or indole, may be performed ona membrane fraction from cells expressing the polypeptides describedhere.

The following examples are illustrative only and are not meant to limitthe scope of invention as set forth in the Summary, Description or inthe Claims.

EXAMPLES Example 1

Screening of Mouse Olfactory Sensory Neurons to Identify Receptors thatBind Indole and/or Skatole

The olfactory epithelium containing native olfactory neurons from amouse was isolated and dissociated into single cells and thentransferred to a dissociation buffer for a gentle mechanical andenzymatic dissociation. In order to optimize the dissociation protocol,the general procedure for making fresh dissociation buffer was followedas set forth in Araneda et al. (2004), A Pharmacological Profile of theAldehyde Receptor Repertoire in Rat Olfactory Epithelium. Journal ofPhysiology: 555:743-756 with the following modifications: DNAse Iinstead of DNAse II, Ca²⁺ concentration elevated to 5 mM and pH adjustedat 7.35 at 32°. Dissociated neurons were then seeded on a coverslipallowing the screening of >2,500 cells by fluorescence microscopy. Thecells were then loaded with a calcium sensitive dye (Fura-2 AM) for 30minutes at 31° C. and transferred onto a microscope ready for screening.Dissociated olfactory neurons were screened as previously described(Malnic et al., 1999; Araneda et al., 2004), but with a motorizedmovable microscope stage to increase the number of cells that can bescreened. The cells were then stimulated by perfusing diluted solutionsof odorants (in physiological saline) over the dissociated olfactoryneurons.

Olfactory neurons that responded to indole and skatole were identifiedby stimulating receptors with 50 μm indole followed by 50 μm skatole andmonitoring the intracellular Ca²⁺ flux indicated by changes in Fura-2fluorescence. FIG. 2 displays Ca²⁺ imaging traces for 6 independentolfactory sensory neurons specifically activated by both indole andskatole (50 μM each). All cells were also stimulated with 20 μMforskolin (fork), a pharmacological activator of the enzyme adenylatecyclase ACIII, to confirm viability and neuronal identity of the cells.Timescale units on the X axis, 6 seconds frame. Average FluorescenceIntensity, relative fluorescent unit as a result of a ratiometric340/380 nm recordings.

Example 2

Isolation and Amplification of mRNA from Indole and Skatole SensitiveCells to Generate cDNA Sequences of the “Malodor Receptor” Gene for theIndole and Skatole Sensitive Receptors

The activated olfactory sensory neurons of Example 1 were isolated usinga glass suction micropipette and then pooled into one sample forsubsequent identification of the odorant receptor genes expressed asmRNA in the responding cells. Malodor receptor gene sequences wereidentified from indole and skatole-sensitive cells as follows: Four setsof more than five (5) olfactory neurons (6, 10, 15, and 15 cells each)activated by one or more malodor compounds were isolated using a glassmicroelectrode attached to a micromanipulator. Micropipettes wereengineered from Borosilicate glass (Item # B150-86-10, SutterInstruments) and pulled using a microelectrode puller (Model P-1000,Sutter Instruments) under the following conditions: Heat, ramp temp+200C; Pull=0; Vel=120; Pressure=500. Pulled pipette tips were brokenmanually and secured onto a Newport micromanipulator (model MW3R) forisolating olfactory sensory neurons activated by indole and skatole asdetermined in a calcium imaging assay. Activated OSNs were isolated bygentle suction applied to the back-end of the micropipette.

The mRNA of neurons was purified and amplified according to the Eberwinemethod (MessageAmp II aRNA kit—Ambion, AM1751). Pooled neurons wereheated to 75° C. for 10 minutes in Resuspension Buffer (SuperScript IIICells Direct cDNA system, 46-6321-Invitrogen) to break the cell membraneand to render their mRNA available for amplification. A linearamplification according to the Eberwine method (in vitro transcription)ensures the maintenance of the relative transcription levels ofexpressed genes. First-strand cDNA was obtained according to the Ambionkit with a incubation for 2 h at 42° C. Second strand cDNA synthesis wasperformed using the same kit after a second incubation for 2 h at 16° C.Doubled stranded cDNA was used as a template to generate thecorresponding cRNA (14 h incubation at 37° C.), which is then purified.These cRNA synthesis steps were repeated for a second IVT. The twoconsecutive overnight (14 h) rounds of in vitro transcription yieldedsufficient amounts of cRNA; Amplified cRNA was then used to generate anIllumina HiSeq cDNA library. The sequence of the entire transcriptomewas then obtained by Next-Generation Sequencing (NGS).

The identity of the mouse malodor, specificallyindole and/or skatole,olfactory receptors was determined by comparing the NGS results to areference genome sequence of the same species. For this, the resultingshort sequences of 150 base pairs (commonly referred to as reads)derived from NGS were aligned against the reference genome of the mouse(UCSC version mm9 or mm10) in order to build the full transcriptome ofthese cells. Quantitative analysis of the transcriptome data yielded alist of transcribed odorant receptor genes and their respectiveexpression levels. Odorant receptor genes that showed abundant levels ofmRNA (abundant reads) or were present in more than one replicateexperiment were considered candidate indole and skatole receptors andare set forth in FIG. 3, which summarizes representatives NGS resultsfrom 4 independent skatole/indole cell picking experiments. The barsrepresent candidate indole/skatole OR genes with the most abundant mRNAsthat were retained after visual inspection. The Y axis represents themRNA abundance levels normalized to the level of OMP mRNA, accountingfor the number of OSNs picked per sample. Star (*), receptors identifiedin more than one experiment. OMP (Olfactory Marker Protein) is aspecific biomarker for mature olfactory neurons. By coupling calciumimaging and NGS as described above the following 5 receptor sequenceswere identified as putative mouse indole and skatole ORs: Olfr665 (SEQID NO: 9 and SEQ ID NO: 10), Olfr740 (SEQ ID NO: 1 and SEQ ID NO: 2),Olfr741 (SEQ ID NO: 3 and SEQ ID NO: 4), Olfr743 (SEQ ID NO: 5 and SEQID NO: 6), and Olfr745 (SEQ ID NO: 7 and SEQ ID NO: 8.

Example 3 Identification of the Mouse and Human Malodor Receptors

The identification of a subset of 5 mouse indole and skatole ORs among˜1,200 ORs in the mouse genome by coupling calcium imaging and NGS(Examples 1-2) enabled the rapid identification of additional mouse andnew human indole and skatole ORs by in silico methods. Since ORs withsimilar overall amino acid identity are likely to display similarodorant response profiles [Adipietro et al. (2012)], a list of candidateindole and skatole ORs was expanded by querying public mouse and humangenome databases with the mouse OR sequences derived from NGS analysisof indole/skatole sensitive olfactory sensory neurons isolated bycalcium imaging. The identity of the mouse malodor olfactory receptorswas determined by comparing the results of the NGS to a reference genomesequence of the same species. The putative malodor receptors will be themost highly abundant mRNA in the olfactory neuron-derived NGS sample orpresent in more than one independent biological replicate, but not in acontrol sample lacking olfactory neurons that respond to the malodor(s).Standard bioinformatics tools were used to identify the most closelyrelated human odorant receptor(s) to the putative mammalian (non-human)malodor receptor(s) under the assumption that homologous sequencereceptors retain similar function (Adipietro et al. (2012)). Defaultparameters of BLASTP and/or BLASTN algorithm were used. Table 1 liststhe identified mouse odorant receptors (OR) and the predicted humanorthologs.

TABLE 1 NGS Identified Mouse ORs Human Orthologs Amino Acid IdentityOlfr665 OR52N2 77% (SEQ ID NO: 10) (SEQ ID NO: 12) Olfr740 OR11G2 77%(SEQ ID NO: 2) (SEQ ID NO: 14) Olfr741 OR11G2 73% (SEQ ID NO: 4) (SEQ IDNO: 14) Olfr743 OR11G2 75% (SEQ ID NO: 6) (SEQ ID NO: 14) Olfr745 OR11H682% (SEQ ID NO: 8) (SEQ ID NO: 16)

The related human protein sequences are within at least 60% sequenceidentity to the NGS identified mouse OR sequences listed in Table 1.Identity levels are given relative to the amino acid sequence pairwisecomparison after BLASTP algorithm. Twenty six (26) new human and mouseORs were identified through BLASTP queries of public genome databases(default parameters). Sixteen (16) were closely related to theNGS-derived genes Olfr740 (SEQ ID NO: 1 and SEQ ID NO: 2), 741 (SEQ IDNO: 3 and SEQ ID NO: 4), 743 (SEQ ID NO: 5 and SEQ ID NO: 6), 745 (SEQID NO: 7 and SEQ ID NO: 8) and define the 740 family. Ten (10) wereclosely related to the NGS-derived receptor Olfr665 (SEQ ID NO: 9 andSEQ ID NO: 10) and define the Olfr665 family. FIGS. 4A and 4B depict thephylogenetic relationships of candidate mouse indole and skatole ORgenes and their predicted human orthologs belonging to the Olfr740 (FIG.4A) and Olfr665 (FIG. 4B) receptor families. The relationships wereobtained by protein sequence-based Neighbor-Joining phylogenetic treereconstruction. Corresponding human orthologs are indicated by arrows.Human OR gene names annotated as pseudogenes are marked with a ‘P’.Pseudogenes are likely non-functional. OR11H7P is a segregatingpseudogene (SP) with known functional alleles. Diamonds (⋄) designatereceptors initially identified by NGS. To determine the amino acididentities for additional candidate mouse and human indole and skatoleORs, the full length protein sequences were manually aligned based onhighly conserved amino acid motifs across the entire OR repertoire(Bioedit sequence alignment tool, version 7.0.5.3). FIG. 8 displays thepairwise amino acid identities for the mouse Olfr740 family and theirpredicted human orthologs. FIG. 9 displays the pairwise amino acididentities for the mouse Olfr665 family and their predicted humanorthologs.

Example 4 Indole and Skatole Activity of Mouse ORs Olfr743, Olfr746, andOlfr740 in HEK293T Cells.

The activity of candidate mouse receptors Olfr740 (SEQ ID NO: 1 and SEQID NO: 2), Olfr743 (SEQ ID NO: 5 and SEQ ID NO: 6), and Olfr746 (SEQ IDNO: 37 and SEQ ID NO: 38) to both indole and skatole malodors wereconfirmed in cell-based cAMP assays. In FIGS. 5A and 5B, FLAG Rho-tagged(SEQ ID 18) Olfr743 (SEQ ID NO: 6) and Olfr740 (SEQ ID NO: 2) cDNAsequences were co-transfected with Gα_(olf). (SEQ ID NO: 21) and exposedto increasing concentrations of indole (left) or skatole (right). FIGS.5C and 5D are repeat experiments showing similar activity of Olfr740(SEQ ID NO: 2) with Olfr743 (SEQ ID NO: 6) with indole and skatole. InFIG. 5E, the FLAG Rho-tagged (SEQ ID 18) Olfr746 (SEQ ID NO: 38) cDNAsequence was co-transfected with Gα_(olf). (SEQ ID NO: 21) and exposedto increasing concentrations of indole or skatole. Odorant-inducedactivity was detected by measuring cAMP levels in the cytosol using theHTRF approach (CisBio kit). A dose-dependent increase of receptoractivity is shown for all three receptors to both molecules.Co-transfection of HEK cells with Gα_(olf)(SEQ ID NO: 21) alone (noreceptor) were used as controls for non-specific activity (control).Activity is defined as the baseline corrected HTRF ratios normalized tothe highest signal in the experiment. The results are displayed in FIGS.5A to 5E. All three receptors show specific dose dependent activity tothe malodor compounds and, as such, validate the process for identifyingreceptors for these compounds. Mouse receptors Olfr740 (SEQ ID NO: 1 andSEQ ID NO: 2) and Olfr743 (SEQ ID NO: 5 and SEQ ID NO: 6) wereidentified from isolated olfactory neurons that responded to both indoleand skatole. Mouse receptor Olfr746 (SEQ ID NO: 37 and SEQ ID NO: 38)was identified in silico by database mining using Olfr740 (SEQ ID NO: 2)and Olfr743 (SEQ ID NO: 6) as query sequences. The receptors aremodified at their N-terminus with the FLAG tag and the first 20 aminoacids of the bovine rhodopsin receptor (SEQ ID 18), transientlyexpressed in HEK 293T cells, and stimulated separately with indole andskatole to confirm their identity as bona fide indole/skatole receptors.Co-expression of the human G alpha subunit Gα_(olf)(SEQ ID NO: 21)activates the Gs transduction pathway that leads to an internal cAMPincrease upon binding to the appropriate ligand. The results confirm theidentity of Olfr740 (SEQ ID NO: 1 and SEQ ID NO: 2), Olfr743 (SEQ ID NO:5 and SEQ ID NO: 6), and Olfr746 (SEQ ID NO: 37 and SEQ ID NO: 38) asindole-skatole receptors.

Example 5 Indole and Skatole Activity of Human OR52N2, OR11G2, OR5AC2,OR4C15, OR8S1, OR11H6, and OR11H4 and Mouse Olfr665 and Olfr740 inHEK293T Cells.

The activity of candidate human indole and skatole malodors receptorsOR52N2 (SEQ ID NO: 11 and SEQ ID NO: 12), OR11G2 (SEQ ID NO: 13 and SEQID NO: 14, OR5AC2 (SEQ ID NO: 75 and SEQ ID NO: 76), OR4C15 (SEQ ID NO:79 and SEQ ID NO: 80), OR8S1 (SEQ ID NO: 83 and SEQ ID NO: 84), OR11H6(SEQ ID NO: 15 and SEQ ID NO: 16) and OR11H4 (SEQ ID NO: 49 and SEQ IDNO: 50) and candidate mouse indole and skatole malodors receptorsOlfr665 (SEQ ID NO: 9 and SEQ ID NO 10) and Olfr740 (SEQ ID NO: 1 andSEQ ID NO 2) were confirmed in cell-based calcium-flux assays (FIGS. 6Ato 6J). Cells stably co-expressing Rho-tagged human OR52N2 (FIG. 6A, B)or OR11G2 (FIG. 6C) and Gα₁₅ (SEQ ID NO: 25) were exposed to increasingconcentrations of skatole or indole. FIG. 6B is a repeat experimentshowing similar activity of OR52N2 with indole and skatole.Odorant-induced OR52N2 or OR11G2 activity was detected by measuring themaximum Calcium 5 dye (Molecular Devices) fluorescence change followingodorant exposure. Activity is defined as the baseline corrected RelativeFluorescent Units (RFU) ratios normalized to the highest RFU in theexperiment in FIG. 6A. Relative Fluorescent Units is used to measure thereceptor activity in FIGS. 6B-J. A dose-dependent increase of receptoractivity was recorded and a corresponding dose-response curve is shownfor both compounds. A cell line lacking a receptor, for example hOR52N2,was used as a control for non-specific activity at high concentrations(‘Indole control’ and ‘Skatole control’). The human receptor OR52N2 (SEQID NO: 11 and SEQ ID NO: 12) was identified because of its sequencesimilarity to mouse Olfr665 (SEQ ID NO: 10), an indole/skatole receptorisolated from olfactory neurons responding to both indole and skatole.The human receptor OR11G2 (SEQ ID NO: 13 and SEQ ID NO: 14) wasidentified because of its sequence similarity to mouse Olfr740 (SEQ IDNO: 2), an indole/skatole receptor isolated from olfactory neuronsresponding to both indole and skatole. The receptors were modified withthe Rho sequence and stably expressed in HEK 293T cells. Furtherexamples show indole and skatole dose-response curves for humanreceptors OR5AC2, OR4C15, OR8S1, OR11H6, OR11H4 and mouse receptorsOlfr665 and Olfr740 (FIG. 6D-J). The human receptor OR5AC2 (SEQ ID NO:75 and SEQ ID NO: 76) was identified because of its sequence similarityto mouse Olfr207 (SEQ ID NO: 78), a receptor isolated from olfactoryneurons responding to both indole and skatole. The human receptor OR4C15(SEQ ID NO: 79 and SEQ ID NO: 80) was identified because of its sequencesimilarity to mouse Olfr1211 (SEQ ID NO: 82), a receptor isolated fromolfactory neurons responding to both indole and skatole. The humanreceptor OR8S1 (SEQ ID NO: 83 and SEQ ID NO: 84) was identified becauseof its sequence similarity to mouse Olfr257 (SEQ ID NO: 86), a receptorisolated from olfactory neurons responding to both indole and skatole.The human receptor OR11H6 (SEQ ID NO: 15 and SEQ ID NO: 16) wasidentified because of its sequence similarity to mouse Olfr745 (SEQ IDNO: 8), a receptor isolated from olfactory neurons responding to bothindole and skatole. The human receptor OR11H4 (SEQ ID NO: 49 and SEQ IDNO: 50) was identified because of its sequence similarity to mouseOlfr746 (SEQ ID NO: 38), a receptor isolated from olfactory neuronsresponding to both indole and skatole. The mouse receptor Olfr665 (SEQID NO: 9 and SEQ ID NO: 10) was identified directly from NGS data fromisolated olfactory neurons responding to both indole and skatole. Themouse receptor Olfr740 (SEQ ID NO: 1 and SEQ ID NO: 2) was identifieddirectly from NGS data from isolated olfactory neurons responding toboth indole and skatole. Co-expression of the human G alpha subunit Gα₁₅activates the Gq transduction pathway that leads to an internal Ca²⁺increase upon binding to the appropriate ligand. These results serve tovalidate the process disclosed here is useful for the rapid and reliableidentification of mammalian odorant receptors for malodor compounds.

Example 6 Inhibition of Indole Receptor Olfr743 Activity in HEK293TCells.

Once identified using the procedure outlined herein, the resultingmalodor receptor cell lines can be used to screen chemical libraries forcompounds that modulate their activity and possibly human perception.This experiment was performed under the same conditions as in Example 4.Cells transfected with Rho-tagged (SEQ ID NO: 18) Olfr743 (SEQ ID NO: 6)were exposed to increasing concentrations of indole in the presence orabsence of 300 μM 2-actetonaphthone. 2-acetonaphthone inhibits theactivity of Olfr743 (SEQ ID NO: 6) to indole. The results are presentedin Table 2 and FIG. 7. In the presence of the antagonist compound, arightward shift of in the dose-response curve is observed resulting in a4 fold increase in the EC50 to indole. Activity is defined as thebaseline corrected HTRF ratios normalized to the highest signal in theexperiment. These results show that 2-acetonaphthone binds and inhibitsthe activity of the indole receptor.

TABLE 2 Indole + 2- Indole acetonaphthone 300 μM EC50 54 μM 206 μM

What is claimed is:
 1. A method of identifying olfactory receptors thatare activated by an odor and/or aroma compound comprising: a)dissociating an isolated olfactory epithelium containing nativeolfactory neurons into a single cell from a non-human mammal specieswherein each neuron expresses the olfactory receptor; b) loading theolfactory cells with an indicator dye that allows for the measurement ofmalodor receptor binding activity of the olfactory receptors; c)contacting the olfactory receptors with malodor compounds; d) measuringchanges of odorant-induced neuronal activity; e) isolating one or moreolfactory neurons that were activated by the malodor compound; f)isolating or isolating and amplifying the mRNA of the isolated olfactoryreceptors; g) sequencing at least a portion of the entire transcriptomeof the mRNA by Next-Generation Sequencing; and h) identifying a group ofmalodor olfactory receptors by comparing the sequence of thetranscriptome to a reference genome sequence of the same species.
 2. Themethod of claim 1, further comprising identifying a human receptor thatis most closely related to a malodor olfactory receptor identified inclaim
 1. 3. The method of claim 1, further comprising modifying a cellline to express at least one malodor olfactory receptor identified inclaim
 1. 4. The method of claim 2, further comprising modifying a cellline to express at least one human receptor identified in claim
 2. 5.The method of claim 3, further comprising contacting the malodorolfactory receptor with a test compound to determine if the testcompound binds to the malodor olfactory receptor.
 6. The method of claim4, further comprising contacting the human receptor with a test compoundto determine if the test compound binds to the receptor.
 7. Anexpression vector comprising a) a nucleic acid comprising a nucleotidesequence having at least 90%, 95%, 98%, or 100% sequence identity to SEQID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ IDNO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 27, SEQ ID NO: 29, SEQID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39,SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO:49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ IDNO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77,SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, or SEQ ID NO: 85; or b) anucleic acid encoding a polypeptide comprising an amino acid sequencehaving at least 90%, 95%, 98%, or 100% sequence identity to SEQ ID NO:2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO:12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 28, SEQ ID NO: 30, SEQ IDNO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50,SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO:60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ IDNO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, or SEQ ID NO:
 86. 8. A cellthat is recombinantly modified to express an olfactory receptorpolypeptide comprising an amino acid sequence having at least 90%, 95%,98%, or 100% sequence identity to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO:6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO:16, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ IDNO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54,SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO:64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ IDNO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQID NO: 84, or SEQ ID NO: 86; or that comprises the expression vector ofclaim
 7. 9. The cell of claim 7, wherein the cell is selected from thegroup consisting of HEK293, CHO, Xenopus oocytes, COS, yeast, bacteriaand cells derived from the olfactory placode.
 10. A method foridentifying a compound that blocks, inhibits, modulates, and/or enhancesthe activity of an olfactory receptor selected from the group consistingof odorant or aroma receptors that is activated by a compound comprisingindole and/or skatole comprising a) culturing at least one cell of claim8 under conditions conducive to expressing at least one olfactoryreceptor; b) contacting a compound with the at least one receptor; c)measuring the extent to which the compound blocks, inhibits, modulates,or enhances the activity of the receptor by measuring the response ofthe olfactory receptor in the presence and absence of the compound. 11.The method of claim 10, further comprising identifying a compound thatblocks, inhibits, modulates, or enhances the response of the olfactoryreceptor on the basis of the response that was measured in the presenceand absence of the compound; and selecting the identified compound as acompound that blocks, inhibits, modulates, or enhances the response ofthe olfactory receptor.
 12. The method of claim 10, wherein theolfactory receptor polypeptide comprises the amino acid sequence of SEQID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 28, SEQ ID NO: 30,SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO:40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ IDNO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68,SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO:78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, or SEQ ID NO: 86.